MXPA97003256A - Human vastago cell factor, a variantede union of the same and its use farmaceut - Google Patents

Abstract

SCF, which includes the following C-terminus: Glu lle Cys Ser Leu Leu lle Gly LEu Thr Ala Tyr Lys Glu Leu Ser Leu Pro Lys Arg Lys Flu Thr Cys Arg Ala lle Gln His Pro Arg Lys Asp, or a sequence C -terminal, which is substantially homologous to it and its use in medicine, particularly to ensure the proper development of pre-implantation embryos

Description

HUMAN VASTAGO CELL FACTOR. A VARIANT OF UNION OF THE SAME AND ITS PHARMACEUTICAL USE DESCRIPTION OF THE INVENTION The present invention relates to a novel human stem cell factor (SCF) protein, to DNA sequences encoding this protein, to its use in therapy, particularly in in vitro fertilization, as well as to pharmaceutical formulations comprising said protein . Successful implantation of embryos requires the correct development of the pre-implantation embryo, resulting in an incubated blastocyst, which is capable of being implanted into the receptive devil. A considerable group of data have been gathered, which support the idea that soluble growth factors, if secreted by the uterine epithelium, act directly on the embryo to control this procedure (Pampfer, S. et al., Bioessays, 13). : 535-540 (1991), Tartakousky, B., and Ben Yair, E., Development Biology, 146: 345-352 (1991), Anderson, ED, J. Cellular Biochem., 53: 280-287 (1993) and Schuitz, GA and Hevner, S., Mutat, Res., 296: 17-31 (1992)). In addition, developing embryos have been shown to produce a variety of cytokines, which can act in an autocrine fashion on the endometrium to influence their receptivity. Examples of growth factors that are shown to be produced by human embryos include IL-1, IL-6, CSF-1 and TNF-a (Zolti et al., Fertil. Steril., 56 (1991) 256-272 and Witkin et al. J. Reprod. Immunol., 19 (1991) 85-93 It has been shown that TNF-a is present in the culture medium of human embryos up to the morula stage, but not that of the blastocyst (Lachappelle et al., Human Reproduction 8: 1032-1038 (1993)) The production of cytokines by the embryo, therefore, can be regulated in a specific manner in the stage.Data of possible direct effects of cytokines in embryos have been obtained mainly in mouse experiments, where many cytokines have been shown to affect the development of pre-implantation embryos, in vitro.IFN-? and CSF-1, physiological concentrations, inhibit the number of developing embryos in the blastocyst stage (Hill and others, J. Immunol; 139 (1987) 2250-2254). It has also been shown that TNF-a It has more subtle effects. Although TNF-a has no apparent effect on the rates of blastocyst formation, it appears to specifically inhibit the proliferation of cells that contribute to the inner cell mass (ICM), which results in blastocysts with a reduced ICM (Pampfer et al. others, Endocrinology, 134: 206-212 (1994)). Other growth factors also have specific effects on ICM cells. For example, insulin-like growth factors 1 and 2 stimulate ICM proliferation, whereas leukemia inhibitory factor (LlF) inhibits their differentiation (Harvey et al., Mol. Reprod. Dev., 31 (1992) 195 -199). It has been observed, in mouse systems, that embryos cultured in vitro retard development compared to controls in vivo, and exhibit lower pregnancy regimens after embryo transfer (Bowman, P. McLaren, A., J. Embryol, Exp. Morphol., 24: 203-207 (1970)). In this way, a better understanding of the role of growth factors in development can lead to improved in vitro culture conditions and improve the outcome in human IVF programs. The stem cell factor (SCF) is a growth factor structurally related to CSF-1, and acts through the receptor of c-cit-tyrosine kinase. The bone marrow, SCF and CSF-1 act synergistically to promote the proliferation and differentiation of stem cells into macrophage colonies. EP-A-0423980 describes the nucleic acid sequence of human SCF, and discusses the potential uses of SCF under conditions that require the stimulation of cell proliferation, particularly blood cells. In mice, c-cit has shown that it is expressed throughout the pre-implantation development (Arceci et al. (1992)). Now it has been shown that it is true in human embryos. In certain stages, human embryos also express SCF mRNA, suggesting that this growth factor can act in an autocrine fashion. This is in contrast to the mouse, where no expression of SCF was detected in pre-implantation embryos (Arceci et al. (1992)). Transcription of full length SCF consists of eight exons (Martin, F. H. et al., Cell, 63: 203-211 (1990)), said document also discloses a variant form of SCF. A variant binding of SCF has also been described, which arises by virtue of the loss of exon 6 (Flanagan et al., Cell, 63: 1025-1035 (1991)). A novel, additional binding variant has now been found, which seems to arise due to the inclusion of a novel exon consisting of 155 base pairs between exons 3 and 4. This also results in a frame change, and encodes for a species of SCF comprising 33 novel amino acids that follow exon 3, before ending in a frame stop codon, which now appears in exon 4 due to frame change. In this manner, the present invention provides SCF which includes the following C-terminal sequence: Glu Me Cys Ser Leu Leu lie Gly Leu Thr Ala Tyr Lys Glu Leu Ser Leu Pro Lys Arg Lys Glu Thr Cys Arg Ala lie Gln His Pro Arg Lys Asp or a sequence that is substantially homologous to it. Preferably, the novel SCF of the invention comprises the first 39 amino acids of full length SCF (not including any signal sequence), followed by the 33 new amino acids, specified above. In one embodiment, the novel SCF of the invention has a sequence at positions 1-39 substantially homologous to that shown in Figure 2. At the amino acid level, one protein sequence can be considered as substantially homologous to another protein sequence, yes a significant number of the constituent amino acids exhibit homology. At least 40%, 50%, 60%, 70%, 80%, 90%, 95%, and even 99%, in order of preference increase, of the amino acids, can be homologous. In this way, the alternative binding mechanism can result in the production of a novel SCF in human embryos. Therefore, the novel SCF of the invention can be used in the treatment of pre-implantation embryos to ensure correct differentiation and development prior to implantation in a subject. In addition, the invention also provides a DNA sequence encoding a protein of the invention, said sequence includes a sequence substantially homologous to: GAA ATC TGT TCA TTG TTG ATA GGG CTG ACG GCC TAT AAG GAA TTA TCA CTC CCT AAA AGG AAA GAA ACT TGC AGA CGA ATT CAG CAT CCA AGG AAA GAC TGA and includes all other nucleic acid sequences, which, by virtue of the degeneracy of the genetic code, also code for the given amino acid sequence or, which are substantially homologous to said sequence. Sequences that have substantial homology can be considered as those that will hybridize to the nucleic acid sequence shown in Figure 2 under severe conditions (eg, from 35 to 65 ° C in a salt solution of approximately 0. 9 M). DNA constructs comprising DNA sequences of the invention form another aspect of the invention. As discussed herein, the protein of the invention is useful for the treatment of embryos to ensure proper development prior to implantation. It has been shown that SCF acts by binding to the transmembrane receptor c-cit. In addition, it has been shown that human embryos express c-cit through most stages of pre-implantation embryo development. Thus, in additional aspects, the invention provides: (a) a method for ensuring the correct development of a pre-implantation embryo, which comprises the step of administering the SCF of the present invention to a pre-implantation embryo (and preferably a human embryo); and (b) a method for ensuring the correct development of a pre-implantation human embryo, which comprises the step of administering the SCF to a pre-implantation human embryo. In this method, the SCF used can be any of the forms of natural existence, including the variants previously described (Martin et al., Supra and Flanagan et al., Supra), as well as the novel variant described herein. In addition, the invention also provides the use of SCF in the manufacture of a medically to be used to ensure proper development in pre-implantation human embryos. Again, any form of SCF can be used to produce a medically suitable one. The mechanism is preferably presented in the form of a pharmaceutical formulation comprising the protein of the invention together with one or more pharmaceutically acceptable carriers and / or excipients. Said pharmaceutical formulations form one more aspect of the present invention. Such pharmaceutical formulations represent a form in which the SCF can be used in the methods described above. The invention will now be described through the following examples, which should not be construed as limiting the present invention. The examples refer to the following drawings which show: Figure 1, the novel exon sequence and the predicted amino acid sequence; Figure 2, the sequence of human SCF; Figure 3 An agarose gel showing the products of the nested RT-PCR amplification on RNA from human embryos, each panel showing the amplification products with specific primers for different cDNA targets. The amplified cDNAs of the different embryos were loaded in each lane. The lanes are marked according to the cDNA tags in Table 1 (below). Additional samples were lane p, first trimester trophoblast; lane q, 200-cell BeWO cDNA; lane r, 10 ng of human genomic DNA; and lanes, no input cDNA, as a negative control. DNA molecular weight markers were on a scale of 123 base pairs loaded in lane y. The sizes of the expected PCR products are shown in bp.

TABLE 1 Human embryo cDNAs and stage name development controls a 2 cells b 3 cells c 4 cells d 6 cells e 8 cells f morula g blastocyst h culture supernatant of ag j 3 deposited blastocysts k culture supernatant for j I 2 x 6 cells and 1 x 8 cells m culture supernatant for I n 1 x 4 cells and 1 x 6 cells or culture supernatant for n the samples are from the same donor. Figure 4, the primers used for RT-SC, pain A and B external, pain C and D internal.

EXAMPLE 1 Embryo Culture and RN Extraction In this study, cryopreserved human embryos were used, which were fertilized as part of an IVF program. These embryos were donated for search purposes, by the parents, and this study met the requirements of the Human Embryology and Fertilization Authority (Authority of Human Embryology and Fertilization), and the local ethics committee. Frozen embryos were thawed and cultured in a balanced salt medium, Earles, supplemented with 0.4% human serum albumin (Armor Pharmaceuticals UK), until the required development stage, then frozen by flash-mist in liquid nitrogen in 5 μl of the culture fluid (and thus were used through ice crystals).

An identical volume of culture supernatant was frozen as a control. During routine management, the remaining accumulation cells were removed. Total RNA was isolated from the first trimester trophoblast through the method of Chomsczynski and Sacchi, Anal. Biochem., 162: 156-159 (1987), where the frozen tissue was homogenized in 5 ml of pH buffer containing 4 M guanidinium thiocyanate (Gibco BRL Livingston, Scotland), 25 mM sodium citrate pH 7.0, 0.5% sarcosil and 0.1 M 2-mercaptoethanol. The lysate was acidified by the addition of 0.5 ml of 2 M sodium acetate pH4, and phenol-chloroform extracted using 5 ml of phenol saturated with pH regulator and 1 ml of chloroform-isoamyl alcohol (49: 1 v / v) . The suspension was placed on ice for 15 minutes and centrifuged at 10,000 g for 20 minutes at 4 ° C. The aqueous phase containing the RNA was precipitated, washed twice in 70% ethanol, dried and resuspended in TE (10 mM Tris-HCl, pH 7.4 and 1 mM EDTA) The concentration of RNA was determined spectrophotometrically at 260 nm RNA from individual human embryos was prepared using a descending scale protocol based on the above procedure To assist in RNA precipitation, 100 μg of vehicle yeast tRNA was added to the homogenization step (Gibco BRL, Livingston , Scotland) .The remaining details are as described above, except that all volumes were 50 times less, and the entire procedure was performed in 400 μl Eppendorf tubes.

EXAMPLE 2 Reverse Transcriptase-Phenimerase Chain Reaction (RT-PCR) cDNA from half of the total RNA of each embryo was synthesized using AMV reverse transcriptase (Super RT, HT Biotech, Cambridge, UK). 3-5 micrograms of RNA were initiated with oligo dT (Pharmacia), according to the manufacturer's instructions for 60 minutes at 42 ° C. PCR amplification of the cDNA preparations was performed as previously described (Sharkey, A. et al., Molecular Endocrinol., 6: 1235-1241 (1992)), with a Hybaid Omnigene DNA thermal cycler, in a final volume of 30 μl using 1 U of Taq DNA polymerase (Cetus, Emeryville, CA) and '10 μM of each pair of external primers (see Figure 4) in the pH regulator recommended by the manufacturer. The following cycle profile was used: -30s at 95 ° C, 30s at X ° C, 30s at 72 ° C for 30 cycles, where X is the softening temperature for each pair of cytokine initiators, as shown continuation.

External Initiators (° C) Internal Initiators (° C) SCF 54 54 HistRNA 52 59 c-cit 56 56 Oligonucleotide primers Oligonucleotide primers for SCF, c-cit and synthetase of histidyl-t RNA were synthesized on a DNA synthesizer, Cruachem PS250. Initiator sequences were designed from the published nucleotide sequences (see Figure 4), so that the amplification of any genomic DNA contamination can result in a differentially sized product of the cDNA species. Due to the small amount of material, two pairs of primers were used for each target cDNA, in a nested PCR protocol. We amplified 1/30 of the cDNA products using Amplitaq (Cetus), in the pH regulator recommended by the manufacturers. After 30 cycles of PCT using the external initiator pair, 1/5 of the first round reaction was transferred to a fresh tube containing the first pair of internal initiator, and subjected to an amplification of 30 additional turns. As a negative control, an equal volume of culture fluid, in which the embryo was developed, was extracted and subjected to RT-PCR in the same manner. Also, 200 cells of the BeWo cell line (ECACC No. 86082803) were extracted as a positive control. The primers used in this study are as shown in Figure 4, along with the expected product size. The identity of each product was confirmed through cloning and sequencing as previously described (Sharkey et al., Mol.Endocrinol. (1992)). To ensure that the detected product resulted from the amplification of cDNA instead of genomic DNA contamination, primers were chosen to cross the intron / exon boundaries. Also, 10 nanograms of genomic DNA were subjected to PCR at the same time as the cDNA, to verify that no product of the expected size was obtained from the genomic DNA.

RESULTS The RT-PCR technique was applied to the total of RNA extracted from human embryos produced in vitro by fertilization. The embryos were cultured in the appropriate stage, then quickly frozen in liquid nitrogen. The stored embryos were thawed and the total RNA was extracted. In order to produce a detectable RT-PCR product, from the total RNA extracted from a single embryo, a nested PCR protocol was employed, wherein the cDNA was subjected to two PCR amplification groups with an external initiator pair, followed by an internal pair. The primers were based on the published cDNA sequences and were designed to cover the limits of intron-exon, so that the genomic DNA amplification of contamination could be easily distinguished from the cDNA products. Initially, the cDNA of each embryo was tested with primers for histidyl-tRNA synthetase (HistRS) to confirm successful RNA isolation and reverse transcription. The primers used gave rise to weak products of more than 400 bp of the genomic DNA, and 110 bp of the cDNA derived from HistRS mRNA. Transcripts for Hist RS were detected in the mRNA of embryos at all stages of development, as well as in decidua and the BeWo cell line of choriocarcinoma, used as positive controls (Figure 3, lanes p and q, respectively). No product was detected in an equal volume of the supernatant of the embryo culture extracted and subjected to RT-PCR, in the same manner, indicating that there was no contamination of the culture with foreign cDNA or RNA. Figure 3 shows examples of similar RT-PCR analysis with primers for SCF and c-cit. The cDNA supplies were reverse transcribed from each RNA sample twice separately, and the PCR assays were repeated twice in each cDNA delivery. The results are shown in Figure 3, which exhibits the expression pattern of c-cit and SCF during the development of the pre-implantation. The identity of the PCR fragments of the correct size was determined by sequencing the cloned PCR products. In cases where novel dimensioned products were observed, they were also cloned and sequenced. For SCF the predicted fragment is 966 bp. However, SCF transcripts appeared to show stage-specific differences in size. After cloning and sequencing, the new product arose due to an alternative binding case, which inserted a new exon between exons 3 and 4. The predicted sequence of the novel transcript is shown in Figure 1. The binding pattern Novelty also implies a frame change, giving a total of 33 new amino acids, before a frame stop codon in exon 4. In a similar analysis using specific primers for c-cit, the receptor for SCF showed that c-cit it was expressed in most of the stages of human pre-implantation embryo development. This suggests that the embryo has the ability to respond to SCF throughout this period.

DISCUSSION It has been shown that many growth factors influence the development of cultured pre-implantation mammalian embryos (for a review see Anderson, ED, J. Cellular Biochem., 53: 280-287 (1993) and Schuitz, GA and Hevner, S., Mutat, Res., 296: 17-31 (1992)). However, there is good evidence for species-to-species differences in the expression of growth factor receptors in the development of pre-implantation. For example, EGF mRNA is expressed in the pig embryo, but has not been found at any stage in pre-implantation mouse embryos (Vaughan et al., Development, 116: 663-669 (1992); Rapolee et al., Science , 241: 1823-1825 (1988), and Watson, AJ et al., Biol. Reprod., 50: 725-733 (1994)). Therefore, the usefulness of these studies for researchers interested in factors for the control of the development of human pre-implantationIt is limited. In addition, the specific growth factors and receptors, investigated in such studies, have often been chosen on a basis for a particular purpose. For both ethical and practical reasons, this approach is not suitable for use in human embryos. Therefore, a nested RT-PCR method has been used, which allows us to classify the expression of growth factor and receptor mRNA in individual pre-implantation human embryos. This method has been widely used during the last five years in other species, as it is reliable, sensitive and economical in the use of embryonic material. RT-PCR with primers for histatin synthetase I-A R Nt was used in cDNA samples to confirm that the cDNA has been successfully prepared from each sample of embryo RNA. The cDNA specific for this ruling gene was successfully detected in cDNA samples made even from a single 2-cell embryo, indicating that the method was sufficiently sensitive for this study. The SCF was expressed in the 2-cell stage, and then reappeared in the 6-cell stage. This is consistent with maternal expression followed by the re-expression of the embryo genome in the 6-cell stage (Braude, P. et al., Nature, 332: 459-461 (1988)). Transcripts of SCF seem to show specific differences in the stage in the size of the transcript. In cloning and sequencing, these were found due to the alternative binding of the primary transcript. Two of these variants were similar to those previously published (Martin and others, supra, and Sharkey, A. and others, Mol.Endocrinol., 6: 1235-1241 (1992)), and one was a novel way that predicts a species of SCF with 33 new amino acids in the carboxy term. Many variants of SCF are now known, some of which are membrane bound and are bioactive. The species expressed by the pre-implantation embryo include those that are known to be bioactive, and indicate that various forms of SCF can act through c-cit expressed by the embryo, and may affect the development of the embryo at this time.

LIST OF SEQUENCES (1) GENERAL INFORMATION: i) APPLICANT: A) NAME: APPLIED RESEARCH SYSTEMS ARS HOLDING NV B) STREET: 14 JOHN B GORSIRAWEG C) CITY: CURACAO D) STATE: CURACAO E) COUNTRY: NETHERLANDS ANTILLES (F) POSTAL CODE (AREA): NL A) NAME: SHARKEY, Andrew Mark B) STREET: 89A Queen Ediths Way C) CITY: Cambridge (D) STATE: Cambridgeshire E) COUNTRY: United Kingdom 'F) POSTAL CODE (AREA): CB1 4PL A) NAME: SMITH, Stephen Kevin (B) STREET: 14 Hertford Street C) CITY: Cambridge D) STATE: Cambridgeshire E) COUNTRY: United Kingdom F) POSTAL CODE (AREA): CB4 3AG (A) NAME: DELLOW, Kimberley Anne (B) STREET: c / o Dept. Cardiothoracic Surrgery, Imperial College (C) CITY: Dovehoise Street, London (E) COUNTRY: United Kingdom (F) POSTAL CODE (AREA): SW3 6LY (ii) TITLE OF THE INVENTION: THERAPEUTIC PROTEIN (iii) NUMBER OF SEQUENCES: 18 (iv) LEGIBLE FORM IN COMPUTER: (A) TYPE OF MEDIUM: flexible disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) PROGRAM: Patent In Relay # 1.0, Version # 1.30 (EPO) ) (v) CURRENT REQUEST DATA: NO. OF APPLICATION: WO PCT / GB95 / 02547 (vi) PREVIOUS APPLICATION DATA: (A) APPLICATION NUMBER: GB 9422293.2 (B) DATE OF SUBMISSION: 4-NOV.-1994 (2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 33 AMINO ACIDS (B) TYPE: amino acid (C) STRING STRUCTURE: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: Glu lie Cys Ser Leu lie Gly Leu Thr Ala Tyr Lys Glu Leu Ser 1 5 10 15 Leu Pro Lys Arg Lys Glu Thr Cys Arg Ala He Gln His Pro Arg Lys 20 25 30 Asp (2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 102 base pairs (B) TYPE: nucleic acid (C) CHAIN STRUCTURE: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (xi) SEQUENCE DESCRIPTION: ID NO: 2: GAMTCTGTTCApGTTGATAGGGCTGA8GOT ^ 60 AAAGAAACTT GCAGAGCAAT TCAGCATCCA AGGAAAGACT GA 102 (2) INFORMATION FOR SEQ ID NO: 3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 180 base pairs (B) TYPE: nucleic acid (C) CHAIN STRUCTURE: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (ix) ASPECT: (A) NAME / KEY: CDS (B) LOCATION: 1 .114 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: ATG GAT GTT TTG GAA ATC TGT TCA TTG TTG ATA GGG CTG ACG GCC TAT 48 Met Asp Val Leu Glu He Cys Ser Leu Leu He Gly Leu Thr Wing Tyr 1 5 10 15 AAG GAA TTA TCA CTC CCT AAA AGG AAA GAA ACT TGC AGA GCA ATT CAG 96 Lys Glu Leu Ser Leu Pro Lys Arg Lys Glu Thr Cys Arg Ala lie Gln 20 25 30 CAT CCA AGG AAA GAC TGA CAGCTTTGAA AGAGACCTGA TAATGATGCA 144 His Pro Arg Lys Asp * 35 AGTAGGAACT TGCATGTGCT TGAACCAAGT CATTGT 180 (2) INFORMATION FOR SEQ ID NO: 4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 38 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4 Met Asp Val Leu Glu He Cys Ser Leu Leu He Gly Leu Thr Ala Tyr 1 5 10 15 Lys Glu Leu Ser Leu Pro Lys Arg Lys Glu Thr Cys Arg Ala He Gln 20 25 30 His Pro Arg Lys Asp 35 (2) INFORMATION FOR SEQ ID NO: 5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 820 base pairs (B) TYPE: nucleic acid (C) STRING STRUCTURE: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (ix) ASPECT: (A) NAME / KEY: CDS (B) LOCATION: 17..643 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5 AAGCTTGCCT TTCCTT ATG AAG AAG ACA CAA ACT TGG ATT CTC ACT TGC 49 Met Lys Lys Thr Gln Thr Trp He Leu Thr Cys 40 45 ATT TAT Cp CAG CTG CTC CTA TTT AAT CCT CTC GTC AAA ACT GAA GGG 97 He Tyr Leu Gln Leu Leu Leu Phe Asn Pro Leu Val Lys Thr Glu Gly 50 55 60 65 ATC TGC AGG AAT CGT GTG ACT AAT AAT GTA AAA GAC GTC ACT AAA TTG 145 He Cys Arg Asn Arg Val Thr Asn Asn Val Lys Asp Val Thr Lys Leu 70 75 80 GTG GCA AAT CTT CCA AAA GAC TAC ATG ATA ACC CTC AAA TAT GTC CCC 193 Val Wing Asn Leu Pro Lys Asp Tyr Met He Thr Leu Lys Tyr Val Pro 85 90 95 105 110 2895íi.íBs? Ss? Í £ | ß 337 GTG GAT GAC CTT GTG GAG TGC GTG AAA GAA AAC TCA TCT AAG GAT CTA 385 Val Asp Asp Leu Val Glu Cys Val Lys Glu Asn Ser Ser Lys Asp Leu 150 155 160 AAA AAA TCA TTC AAG AGC CCA GAA CCC AGG CTC ITT ACT CCT GAA GAA 433 Lys Lys Ser Phe Lys Ser Pro Glu Pro Arg Leu Phe Thr Pro Glu Glu 165 170 175 pC TTT AGA ATT TTT AAT AGA TCC ATT GAT GCC TTC AAG GAC TTT GTA 481 Phe Phe Arg He Phe Asn Arg Ser He Asp Ala Phe Lys Asp Phe Val 180 185 190 GTG GCA TCT GAA ACT AGT GAT TGT GTG Gp TCT TCA ACA HA AGT CCT 529 Val Wing Ser Glu Thr Ser Asp Cys Val Val Ser Ser Thr Leu Ser Pro 195 200 205 GAG AAA GAT TCC AGA GTC AGT GTC ACA AAA CCA TTT ATG HA CCC CCT 577 Glu Lys Asp Ser Arg Val Ser Val Thr Lys Pro Phe Met Leu Pro Pro 210 215 220 225 GH GCC GCC AGC TCC Cp AGG AAT GAC AGC AGT AGC AGT AAT AGT AAG j 625 Val Ala Ala Ser Ser Leu Arg Asn Asp Ser Ser Ser Ser Asn Ser Lys 230 235 240 TAC ATA TAT CTG Ap TAA TGCATGCATG GCTCCAApA GCACCTATAG_673_Tyr He Tyr Leu He * 245 GAGTApGCA TGGGCTpCA AGGAAACpC TACATTTAp ApApGATA CTGpCTGp 733 ACTGpApc cppATGGT cpcpGAGA cpAAGprG TAGMpAM? TCCCTAGA 793 GCTGGAGATA ATGpTAGAG AApAGG 820 (2) INFORMATION FOR SEQ ID NO: 6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 209 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: Met Lys Lys Thr Gln Thr Trp He Leu Thr Cys He Tyr Leu Gln Leu 1 5 10 15 Leu Leu Phe Asn Pro Leu Val Lys Thr Glu Gly He Cys Arg Asn Arg 20 25 30 Val Thr Asn Asn Val Lys Asp Val Thr Lys Leu Val Ala Asn Leu Pro 35 40 45 Lys Asp Tyr Met He Thr Leu Lys Tyr Val Pro Gly Met Asp Val Leu 50 55 60 Pro Ser His Cys Trp He Ser Glu Met Val Val Gln Leu Ser Asp Ser 65 70 75 80 Leu Thr Asp Leu Leu Asp Lys Phe Ser Asn He Ser Glu Gly Leu Ser 85 90 95 Asn Tyr Ser He He Asp Lys Leu Val Asn He Val Asp Asp Leu Val 100 105 110 Glu Cys Val Lys Glu Asn Ser Ser Lys Asp Leu Lys Lys Ser Phe Lys 115 120 125 Ser Pro Glu Pro Arg Leu Phe Thr Pro Glu Glu Phe Phe Arg He Phe 130 135 140 Asn Arg Ser He Asp Wing Phe Lys Asp Phe Val Val Wing Ser Glu Thr 145 150 155 160 Be Asp Cys Val Val Be Ser Thr Leu Be Pro Glu Lys Asp Ser Arg 165 170 175 Val Ser Val Thr Lys Pro Phe Met Leu Pro Pro Val Ala Wing Ser 180 185 190 Leu Arg Asn Asp Ser Ser Ser Ser Asn Ser Lys Tyr He Tyr Leu He 195 200 205 (2) INFORMATION FOR SEQ ID NO: 7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 17 base pairs (B) TYPE: nucleic acid (C) CHAIN STRUCTURE: individual ( D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: CCGCAGGTCG AGACAGC 17 (2) INFORMATION FOR SEQ ID NO: 8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) STRING STRUCTURE: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: CAAACACCTT CTCGCAA 18 (2) INFORMATION FOR SEQ ID NO: 9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base pairs (B) TYPE: nucleic acid (C) STRING STRUCTURE: individual (D) TOPOLOGY: linear (i¡) TYPE OF MOLECULE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: CTTCAGGGAG AGCGCGTGC 19 (2) INFORMATION FOR SEQ ID NO: 10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) CHAIN STRUCTURE: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: TCATCAGGAC CCAGCTGTGC 20 (2) INFORMATION FOR SEQ ID NO: 11: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) CHAIN STRUCTURE: individual (D) TOPOLOGY: linear (ii) ) TYPE OF MOLECULE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: CAATGCGTGG ACTATCTGCC 20 (2) INFORMATION FOR SEQ ID NO: 12: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRING STRUCTURE: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: GTTCTAAATG AGACCCAAGT 20 (2) INFORMATION FOR SEQ ID NO: 13: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) CHAIN STRUCTURE: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: AACAGTAAA CGGAGTCGCC 20 (2) INFORMATION FOR SEQ ID NO: 14: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) CHAIN STRUCTURE: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: ACAGTGTTGA TACAAGCCAC 20 (2) INFORMATION FOR SEQ ID NO: 15: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) CHAIN STRUCTURE: individual (D) TOPOLOGY: linear (I) TYPE OF MOLECULE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15: GAAGTACAGT GGAAGGTTGT T 21 (2) INFORMATION FOR SEQ ID NO: 16: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) CHAIN STRUCTURE: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16: CATCGGCCAC TAAAGTGTGC T 21 (2) INFORMATION FOR SEQ ID NO: 17: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) CHAIN STRUCTURE: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17: GGTTGTTGAG GCAACTGTT A 21 (2) INFORMATION FOR SEQ ID NO: 18: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRING STRUCTURE: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18: GGTGACCCAA ACACTGATTC 20

Claims (1)

1. CLAIMS 1. - A DNA encoding SCF, which has the following C-terminal amino acid sequence: Glu He Cys Ser Leu Leu He Gly Leu Thr Ala Tyr Lys Glu Leu Ser Leu Pro Lys Arg Lys Glu Thr Cys Arg Ala lie Gln His Pro Arg Lys Asp. 2 - The DNA according to claim 1, wherein the C-terminal sequence starts at position 40 of the amino acid sequence of SCF. 3. The DNA according to claim 2, wherein the sequence in positions 1 to 39, is that shown for positions 1-39 in Figure 2. 4. The DNA according to any of claims 1 to 3, where it includes the following sequence: GAA ATC TGT TCA TTG TTG ATA GGG CTG ACG GCC TAT AAG GAA TTA TCA CTC CCT AAA AGG AAA GAA ACT TGC AGA CGA ATT CAG CAT CCA AGG AAA GAC TGA. 5. The DNA, which hybridizes under severe conditions to DNA as defined in any of claims 1 to 4. 6. A DNA construct comprising the DNA defined in any of claims 1 to 5. 7. - The SCF encoded by DNA as defined in any of claims 1 to 5. 8. The SCF according to claim 7, for use in medicine. 9. The SCF according to claim 8, to be used, to ensure the correct development of pre-implantation embryos. 10. The use of SCF according to claim 6, in the manufacture of a medically to be used, to ensure the proper development of pre-implantation embryos. 1 .- A pharmaceutical formulation comprising SCF according to claim 6, together with one or more pharmaceutically acceptable vehicles and / or excipients. 12. A pharmaceutical formulation according to claim 1, which is used to ensure the proper development of pre-implantation embryos. 13. A method for ensuring the correct development of pre-implantation embryos, which comprises the step of administering to an embryo, the SCF according to claim 6. 14. - A method to ensure the correct development of pre-implantation human embryos, which comprises the step of administering to a human embryo the SCF.